A catalytic hairpin assembly system with sliding replication for the detection of piRNAs

Abstract

As novel noncoding small RNA molecules, piRNAs play crucial roles in cancer development. However, due to their short sequences, easy degradation, and low abundance, developing specific detection methods is challenging. Rapid and early detection is important for the early clinical detection of tumours. Here, a novel one-step, dual-signal amplification piRNA detection system based on sliding replication and catalytic hairpin assembly (CHA), termed CTA, was developed for rapid, ultrasensitive and specific detection of piRNA-823. By utilizing the unique characteristics of tandem repeat sequences to improve amplification efficiency and fluorescence signal intensity, CTA achieved efficient target recognition and signal amplification by embedding tandem repeat sequences in one of the hairpin probes and utilizing chain displacement reactions to produce strong and detectable signals. CTA detected piRNA-823 with a low detection limit of 65.31 fM. Moreover, the whole detection process could be completed within 45 min. In addition, CTA performed excellently in the detection of cell and cancer samples, and its detection results were consistent with those of RT‒qPCR. More importantly, CTA was successfully applied to effectively differentiate between normal individuals and patients with colorectal cancer. These findings suggest its promising application in the diagnosis of cancer.